A cap at the tip of the bacterial flagellum uses a
dynamic differential binding of individual subunits to allow the filament
tip to grow, achieving control of assembly far from the point of protein
translation.

Physical movement is an incredible evolutionary
achievement. Even tiny bacteria such as Escherichia coli and
Salmonella can propel themselves through liquid environments and on
surfaces by the rotation of attached helical appendages called flagella.
The case of the bacterial flagellum might seem
at first glance to be a simple mechanism for self-propulsion, but closer
inspection reveals a sophisticated, self-assembled molecular machine. Now,
in a recent issue of Science, Yonekura et al.1
present a model, based on their electron cryomicroscopy work, that
explains the perplexing aspects of filament assembly.

Flagellum
structureThe flagellum is generally
divided into three structural components (Fig, 1a): (i) a basal body, which is a molecular
rotary motor that includes a drive shaft traversing both the inner and
outer membranes; (ii) a hook, which acts as a flexible joint between the
basal body and the outer filament; and (iii) the filament itself, which is
a long helical structure composed of up to 30,000 polymerized flagellin
subunits2.
A torque generator at the base of the flagellum utilizes
energy available from an electrochemical proton gradient providing the
force necessary to turn the rotor, and propel the organism through a
liquid environment3-5.
In response to environmental cues, flagella rotation is regulated by
accompanying chemosensory machinery to accomplish directed movement6,
7.

Flagellum
assemblyAssembly of the flagellum begins with
the insertion of a ring structure within the cytoplasmic membrane8.
When the cytoplasmic ring structure is completed, it seals off a disc of
membrane into which a secretion apparatus is constructed9.
The secretion apparatus is needed to export flagellar structural subunits
beyond the inner, cytoplasmic membrane. Beyond the cytoplasm, secreted
subunits self-assemble into the growing structure. After hook-basal body
completion and just prior to filament elongation, proteins that make up
the hook-filament junction are added, followed by the cap protein. At this
point, the filament subunits are added between the hook-filament junction
proteins and the
cap. The efficient addition of consecutive filament subunits requires the
cap structure, which stays at the very distal end of the growing filament.
The filament maintains a central channel that is 30 ┼ wide10,
through which partially folded subunits must pass and have the ability to
self-assemble at the tip of the growing structure. Amazingly, the external
filament is 200 ┼ in diameter, yet subunits are added at the distal tip that
can be as far as 15 m away from the entry portal in the cytoplasmic
membrane.

Even though there is clear evidence that the flagellar filament can
self assemble11,
the cap of the flagellar filament serves to control its assembly12.
The cap enables the filament to polymerize with high efficiency, so that
every filament subunit that reaches the tip inserts into place. At the
same time the bacterium continues to secrete other proteins through the
filament that are presumed to simply pass by the cap and out into the
extracellular medium. These include excess hook-filament junction
subunits13,
excess cap14
and a negative regulator of flagellin gene transcription15.
This suggests that the cap acts as a gatekeeper that selectively retains
filament subunits and may even play a role in helping them to fold into
place.

Role of cap in filament polymerizationBefore describing the
mechanism of the capping function, we will first describe the physical
characterization of the cap, and the role of the cap in filament
polymerization in vivo and in vitro that were explained by
the capping model proposed by Yonekura et al.1.
Purified cap forms a decamer in solution, although experimental
observations support the model that the native capping function is
performed by a pentamer16,
17.
Reconstitution of the cap onto filaments reveals that the planar side of
the pentamer is the outer surface, while the disordered terminal regions
of cap subunits are embedded at the filament/cap interface18.
Bacteria that harbor mutations in the cap gene continue to secrete
filament subunits19,
but those subunits do not assemble and instead dissipate into the external
medium (Fig. 1b). If purified filament subunits are added
in sufficient, high concentration to the extracellular medium, the
filament will polymerize onto the hook-filament junction of a cap-less
hook-basal body (Fig. 1c)20.
However, if even one cap monomer is added prior to the addition of
filament subunits, it will block in vitro polymerization of
externally added filament monomers17,
20.
If excess cap is added in vitro, then a functional cap pentamer
will form on the end of the hook-basal body21.
In this case, internally synthesized filament subunits will polymerize
normally, but externally added filament subunits will not (Fig. 1d). This suggests an additional role of the
cap is to effectively increase the local concentration of the filament
subunits that are synthesized in the cytoplasm at the filament-cap
junction. As a result, a nascent, unpolymerized subunit is localized in
proximity to the most recently added subunit to allow it to fold into
place in a thermodynamically favored position at the filament
tip.

The structure of the filament is a spiral arrangement of filament
monomers of 5.5 monomers added per turn while the cap is a planar pentamer of
identical subunits22.
How can the planar cap remain firmly attached to the tip of the elongating
spiral filament and still allow for selective polymerization of filament
subunits and passage of nonfilament proteins between
itself and the growing tip?

A possible answer to this mystery has now come from the electron
cryomicroscopy of the capľfilament complex by Yonekura et
al.1
The cap structure looks like a pentagonal disc with five legs17.
Each leg is differentially attached to the filament subunits at the
filament tip because the cap is planar and the filament end is axial. Yonekura et
al.1
have found that beneath the cap plate a cavity was revealed that is large
enough to allow a newly arrived filament subunit to complete its tertiary
fold just prior to its final quaternary placement in the filament. The
three-dimensional density map of the cap-filament junction also showed the
five sides pertaining to each cap monomer at the junction. In addition,
gaps between the cap plate and the filament end were observed. One of the
five gaps is distinctly larger than the other four at 25 50 ┼. This would be predicted from the axial stagger of
individual filament subunits packed in the filament and the site at which
the authors propose new filament subunits would be added. The addition of
a new filament would force the cap outward to the next most stable
position. This is effectively a step up 4.7 ┼ and over 6.5 ┼. Thus, as a
flagellin subunit is added in the lowest position (the hole), the cap
`walks' along the top of the spiral filament staircase in a direction
opposite to that of filament elongation, taking one `step' with the
addition of each filament subunit (Fig. 2). This model is similar to the action of unscrewing
a lid from a jar, although for the flagellar filament the lid (or cap)
never comes off the jar (the growing filament). The cap would make a
complete rotation with every 55 filament subunits added.

The energy required to move the cap could come from the binding energy
of each newly incorporated filament subunit. This model proposed by
Yonekura et al.1
(and beautifully illustrated at http://www.npn.jst.go.jp/yone.html)
suggests a highly refined mechanism that satisfies all the requirements
for filament self-assembly and represents a novel discovery in structural
design. This mechanism explains how control of assembly can occur even if
it is outside the cell far removed from cellular processes such as
transcription and
translation.